Process for the production of a hybrid structure consisting of coupled silk fibroin microfibers and nanofibers, hybrid structure thus obtained and its use as implantable medical device

ABSTRACT

A method is described for the production of hybrid structures formed by the coupling of nanofibrous parts and microfibrous parts made with silk fibroin, possibly hierarchically organized into complex structures comprising more than two of said parts; these hybrid structures are used as implantable biomedical devices with tailored biological, geometrical and structural features, such that they can be adapted to different application requirements in the field of regenerative medicine.

This application is a national stage application under 35 U.S.C. § 371of PCT Application No. PCT/M2015/058262, filed 27 Oct. 2015, whichclaims priority of Italy Application No. MI2014A001841, filed 27 Oct.2014, which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a process for the production of ahybrid structure consisting of mutually coupled fibroin microfibers andnanofibers, possibly hierarchically organized into complex structurescomprising several primary structures obtained from the coupling ofnano- and microfibrous fibroin; the invention also relates to the hybridstructure obtained by the process, and its use as an implantable medicaldevice for use in a wide range of applications in the field of tissueengineering and regenerative medicine.

BACKGROUND ART

In modern surgery, scaffolds are increasingly used, which areimplantable devices that have the function of temporarily compensatingthe impaired functionality of body parts and tissues, and which are thencolonized by the natural cellular regrowth of the damaged part or tissueto achieve the regeneration of the same.

The materials for producing the scaffolds can be very different,depending on the intended application. For example, inorganic scaffoldsconsisting of mixtures of hydroxyapatite and β-tricalcium phosphate aretypically used for the temporary replacement and regrowth of bonetissue. However, polymeric scaffolds are far more common, preferablybased on biodegradable and biocompatible biopolymers or syntheticpolymers, intended to temporarily replace non-rigid tissues.

A polymeric scaffold must possess a series of surface and bulkproperties optimized for the function to be carried out in vivo. Amongthe properties of interest we may consider the morphologicalcharacteristics at a nano, micro and macroscopic level; thephysical-mechanical properties and performance (ideally, these should beas close as possible to the in vivo characteristics and performance ofthe tissues to be regenerated); and the chemical and biologicalproperties, with particular reference to biocompatibility (i.e. theability of supporting adhesion and cell growth, not causing inflammatoryand/or immunogenic reactions, not releasing hazardous substances, etc.)and to biodegradability or bioresorbability (which must be commensuratewith the residence time of the device in vivo, which in turn depends onthe reconstruction rate of the tissue to be repaired). Other propertiesof interest can be porosity, permeability to fluids, ability to uptake,retain and then release, when required, active agents, growth factors ordrugs, etc. to the implantation site.

A particularly promising natural polymer for use in the production ofscaffolds is fibroin, a silk protein produced in nature by Lepidoptera(domestic species: Bombyx mori; wild species: Antheraea pernyi,Philosamia ricini, etc.), other insects and arachnids. Fibroin can alsobe produced by recombinant DNA techniques. Fibroin is obtained fromnatural silk with the so-called “scouring” treatment, which consists inthe removal of the sericin layer covering the fibroin; this treatment isgenerally carried out through a water bath optionally added with alkalis(soap), acids or enzymes, at temperatures between about 60 and 120° C.,if necessary by operating in an autoclave. The fibroin thus obtained isin the form of microfibers having an average diameter of 12-14 μm, withan ultimate strength of about 600 MPa and elongation at break values of25-30% (values referred to the microfiber of B. mori). Two- orthree-dimensional structures (threads, yarns, woven fabrics, knittedfabrics, nonwoven fabrics, nets, braids, cords, etc.) can be producedwith these microfibers, making use of technologies developed for silkapplications in the textile field and which thus fall within theordinary knowledge of the man skilled in the art.

The interest towards fibroin is mainly due to its provenbiocompatibility, which is expressed through the ability of supportingthe growth and proliferation of various cell types, the lack ofimmunogenic and inflammatory reactions, and the marked angiogenicproperties, particularly useful in the case of the repair/regenerationof living tissues. In addition, the physical-mechanical characteristicsmentioned above allow producing scaffolds with mechanical propertiessuitable for the purpose (in particular, high resistance to tensile,bending, compression stress; good elasticity; resilience, etc.);finally, the scaffolds made of fibroin have biodegradabilitycharacteristics in the medium-long term in vivo (from a few months to1-2 years, depending on the characteristics of the biologicalenvironment of the implantation site), optimal for applications in whichthe scaffold must ensure mechanical support for prolonged times.

Thanks to these properties, fibroin has already been proposed in the artfor the production of scaffolds.

The coupling of polymers in the form of fibers through partialdissolution and subsequent re-deposit of polymer on the fibers is knownfor example from patent application DE 1436311 A1.

U.S. Pat. No. 8,202,379 B1 describes the coupling of fibers of naturalor synthetic polymers by treatment of the same with mixtures containingan ionic liquid and a second liquid compound, generally water, analcohol or a ketone.

These documents only describe the coupling of fibers homogeneous in sizeand chemical, physical and mechanical properties (as derived from thesame production and/or working process).

Patent applications WO 03/043486 A2, EP 2210971 A1 and WO 2011/031854 A1describe fibroin structures intended for the reconstruction of ligaments(in particular the anterior cruciate ligament of the knee), consistingof a hierarchy of fibrous structures assembled at increasing levels upto reaching the dimensions and the mechanical performance required forthe application.

Patent applications WO 2013/012635 A2, WO 2013/082093 A1 and WO2012/111309 A1 describe instead devices based on native fibroinmicrofibers, having a net structure obtained by knitting, optimized forthe reparative surgery of damaged tissues in the abdominal and pelvicareas, for plastic surgery of the breast, and for the realization ofvascular prostheses, respectively.

These scaffolds are produced from fibroin microfibers (which as saidhave diameters of about 12-14 μm) through the use of textiletechnologies, in which the basic silk thread, consisting of at least 20microfibers of fibroin, is generally assembled by doubling and twistingoperations in hierarchically superior structures (“yarns”) whosetransverse dimensions may range from a tenth of a millimeter up to onemillimeter or more. Although the textile structures thus producedusually have excellent characteristics of softness and smoothness and ata macroscopic level they adapt easily to the surface to which theyadhere, at a microscopic level they can display stiffness areas such asto cause local irritation/inflammation reactions; furthermore, due totheir high crystallinity and toughness, fibroin microfibers are able toexert friction forces of such magnitude as to abrade the surface of thetissue with which they come into contact; these problems can lead, inthe worst cases, to the partial or total failure of the implant. Anotherdrawback of fibroin microfiber scaffolds is that they displayunfavorable surface/volume ratios, so that a total area suitable forautologous colonization involves a relatively high load of material tobe placed in the implantation site, with the consequences of a potentialoverload of physiological and metabolic activity due to a localaccumulation of degradation products to be disposed of by the organism.Finally, the natural degradation times of the fibroin microfibers insome cases may be too long compared to the rate of neo-tissue formation,and thus interfere with its growth.

In order to obviate these drawbacks, it has been proposed the use offibroin in the form of nanofibers, that is, having a diameter less thanone micron and typically from a few tens up to a few hundred nanometers.

These nanofibers can be produced by known processes, in which the nativefibroin is first solubilized in a suitable solvent, and then regeneratedwith processes such as force-spinning or electrospinning. In theseprocesses, the solution of fibroin is passed through a capillary tube,called a spinneret, giving rise to a liquid filament of nanoscopicdimensions, which is accelerated towards a collector; in the case offorce-spinning, the acceleration is caused by the centrifugal force (dueto the rotation of the spinneret at a speed of several thousand rpm),while in the case of electrospinning, it is caused by a potentialdifference between the nozzle of the spinneret and a manifold, whichloads the liquid thus causing the production of a jet of solution;thanks to the viscoelastic properties of the polymer, the jet undergoesa drawing process which, accompanied by the simultaneous evaporation ofthe solvent, leads to the production of the nanofiber which accumulateson the collector.

Recent studies have demonstrated the excellent properties of thescaffolds made with fibroin nanofibers.

The article “In vivo regeneration of elastic lamina on fibroinbiodegradable vascular scaffold”, I. Cattaneo et al., Int. J. Artif.Organs 36 (2013) 166, shows that a tubular scaffold of electrospunfibroin, implanted in the abdominal portion of the aorta of the rat,allows the formation of a vascular tissue totally similar to the nativeone from the morphological and functional point of view. The article“Decellularized silk fibroin scaffold primed with adipose mesenchymalstromal cells improves wound healing in diabetic mice”, S. E. Navone etal., Stem Cell Research & Therapy, 5 (2014) 7, shows the effectivenessof electrospun fibroin patches, pre-activated by contact withmesenchymal cells of the adipose tissue, in inducing wound healing indiabetic mice through biological mechanisms involving the directstimulation of angiogenic processes by the material.

Scaffolds have also been described made by coupling of fibroinmicrofibers and nanofibers.

Patent application CN 101879330 A describes a device, proposed as avascular prosthesis and/or a guide for the regeneration of nerves,having a three-layers tubular structure, wherein the inner layer is aporous deposit made from regenerated fibroin, the intermediate layer isa tubular structure of woven microfibers of fibroin in the form of anet, and the outer layer consists of a nanofibrous fibroin structureproduced by electrospinning. The production process of the devicedescribed in this document is complex. The inner layer is initiallyproduced in tubular shape with standard weaving methods, the layer thusobtained is immersed in a solution of fibroin and the resultingintermediate product is dried at 40-60° C. This first intermediateproduct is fitted onto a collector pin for electrospinning, and a layerof nano-microfibrous fibroin is deposited on the outer surface of saidfirst intermediate product mentioned above by means of this technique;the composite thus obtained is then immersed in methanol or ethanol for1-4 hours. Finally, this first composite is introduced into a mold andthe porous layer is produced on its inner tubular surface by depositfrom a fibroin solution; the porosity of the innermost layer is obtainedby a treatment at temperatures between −80° C. and −10° C. The threelayers of this composite are bonded together by means of fibroin filmsthat are formed on the surface of the same during the immersions insolvents or in fibroin solutions. The fibroin films produced accordingto the process of this document, however, once dried and crystallizedare extremely fragile, such as to fracture immediately as they are urgedby tensile, bending, compression, etc., mechanical stress; this can leadto the creation of morphological and mechanical discontinuity betweenthe different layers which can easily lead to a loss of the geometricand performance characteristics, such as the yielding and/or thecollapse of the weaker layers from the mechanical point of view (inparticular the nanofibrous ones).

Patent application CN 102499800 A describes a device which may findapplication as a stent or prosthesis for the repair of small bloodvessels, also in this case consisting of a hybrid structure with threelayers; the inner and the intermediate layer are made of nanofibrousstructures obtained by electrospinning of a fibroin/polycaprolactonemixture, while the outer layer consists of a tubular structure offibroin microfibers, produced with a braiding machine. The three layers,produced separately and fitted one on top of the other as sleeves, areheld together by a series of annular stitches. This device partly hasthe same drawbacks as the former one; furthermore, the fact that thecoupling is realized with stitches spaced apart from one another leavesa partial freedom of movement to the various components; in stressingworking conditions from the mechanical and biological point of view,such as those that can occur in the progress of the implantation invivo, this could create local stresses of such a magnitude as tointerfere with the regenerative processes in progress, especially if thematerial is exposed to flows of physiological fluids.

The object of the present invention is to provide a hybrid compositematerial made of fibroin micro- and nanofibers which allows producingscaffolds for medical applications free from the drawbacks of the priorart.

SUMMARY OF THE INVENTION

This object is achieved with the present invention, which in a firstaspect thereof relates to a process for the production of a hybridstructure made of microfibers and nanofibers of silk fibroin coupled toone another, which comprises the following steps:

-   -   a) preparation of one or more parts made of microfibrous        fibroin;    -   b) preparation of one or more parts made of nanofibrous fibroin;    -   c) treatment of said one or more parts of nanofibrous fibroin        and of said one or more parts of microfibrous fibroin,        separately or after coupling, with a solvent for fibroin and/or        with a solution comprising fibroin dissolved in a solvent;    -   c′) if in step c) the nanofibrous and microfibrous parts have        been treated separately with a solvent for fibroin and/or with a        solution comprising fibroin dissolved in a solvent, coupling of        said parts;    -   d) consolidation of the hybrid microfibrous/nanofibrous        structure obtained in step c) or in step c′) by thermal        treatment at a temperature between 10° C. and 150° C., for a        time between 1 minute and 24 hours;    -   e) removal of the solvent by washing with water or a        water-alcohol mixture or by evaporation at temperatures between        10° C. and 100° C., possibly under vacuum,

wherein the solvent used in step c) is selected from: formic acid,1,1,1,3,3,3-hexafluoro-2-propanol, trifluoroacetic acid,hexafluoroacetone, N-methylmorpholine N-oxide, ionic liquids, calciumchloride-ethanol-water mixtures, calcium nitrate-methanol-watermixtures, aqueous solutions of lithium salts and mixtures among thesesolvents and/or with water.

The process of the invention may further comprise the followingadditional steps:

-   -   f) coupling of two or more micro/nanofibrous hybrid structures        obtained according to steps a) to e) to form a superior        hierarchical structure;    -   g) consolidation of the superior hierarchical structure thus        obtained by repetition on the same of steps c) to e) mentioned        above.

In a second aspect thereof, the invention relates to implantable medicaldevices that use the hybrid structures obtained with said process.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in detail hereinafter with referenceto the accompanying drawings, in which:

FIG. 1 schematically shows an apparatus for carrying out the operationof coupling nanofibrous and microfibrous parts according to theinvention;

FIGS. 2 and 3 show photomicrographs obtained with scanning electronmicroscope (SEM) of samples of hybrid structures produced according tothe invention;

FIG. 4 shows photomicrographs similar to those in FIGS. 2 and 3 obtainedon samples of hybrid structures produced according to the prior art;

FIG. 5 shows the IR spectra of hybrid structures produced according tothe invention compared with samples of fibroin microfibers alone orfibroin nanofibers alone;

FIG. 6 shows graphs of differential scanning calorimetry (DSC) relatingto hybrid structures produced according to the invention and to fibroinmicrofibers alone or fibroin nanofibers alone;

FIG. 7 shows load/elongation diagrams of samples of hybrid structuresproduced according to the invention and of fibroin microfibers alone orfibroin nanofibers alone;

FIG. 8 shows load/elongation diagrams of samples of hybrid structuresproduced according to the prior art;

FIG. 9 schematically shows an apparatus for carrying out peeling testson the layers of hybrid structures;

FIG. 10 shows the graphs of applied force/peeling stroke obtained intests of detachment of the layers of hybrid structures of the inventionand of the prior art;

FIG. 11 show graphs representative of cell growth on medical devicesmade with hybrid structures of the invention; and

FIG. 12 shows histograms relating to genotoxicity measures on patchsamples made with microfibers alone, nanofibers alone and with thehybrid structures made from fibroin of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description and in the following claims, the term “part”means a body formed from homogeneous fibers of fibroin, that is, onlymicrofibers or only nanofibers, while the term “hybrid structure” meansa body formed by coupling of at least one part formed from nanofibersand at least one part formed from microfibers.

The inventors have discovered that by combining fibroin nanofibers andmicrofibers according to the process described hereinafter it ispossible to form hybrid structures provided with mechanical propertiesadapted for the production of implantable medical devices.

The nanofibers useful for the purposes of the invention have diametersbetween 20 nm and 1.5 μm, and preferably between 0.4 and 1 μm.Microfibers instead typically have diameters between about 10 and 15 μm;the microfibers may also be coupled into multifiber yarns, containingeven up to 500 individual silk filaments. In the remainder of the text,the unit of measurement den is also used, typical of the textiletechnology, defined as weight in grams of 9000 meters of fiber or yarn.

In the first aspect thereof, the invention relates to the productionprocess of micro/nanofibrous hybrid structures by coupling of one ormore fibroin microfibrous parts and one or more fibroin nanofibrousparts.

Step a) of the process consists in the preparation of one or moremicrofibrous fibroin parts. The fibroin microfibrous part confers theshape and mechanical strength to the final medical device; as first stepof the process it is therefore necessary to prepare a fibroinmicrofibrous part having shape and dimensions essentially correspondingto those of the desired device. Microfibrous fibroin is used as startingmaterial for the production of this part, in the form of a silk yarnhaving a count of between 10 den and 400 den, and preferably between 15den and 100 den. The silk yarn can be used after scouring or it can beused raw and scoured after the production of the part. The microfibrousfibroin can be added with bioactive agents including growth factors,drugs, cells, antibiotics, antiviral agents, enzymes, vitamins, etc.,for example by means of chemical and/or enzymatic reactions. Thisaddition may be made prior to step a), during any of steps c) to g), orlater on. This part may be formed by one or more elements obtained byany of the techniques known in the textile industry, such as weft-warpweaving (obtaining an orthogonal fabric), production techniques ofnonwoven fabric, knitting, braiding, or the technique known as “filamentwinding”, which consists in the winding of yarns, according to variableinterweaving patterns, around a rotating spindle, which leads to theobtaining of hollow cylindrical structures.

Step b) of the process consists in the preparation of one or morenanofibrous fibroin parts. Nanofibrous fibroin parts useful for thepurposes of the invention can be obtained through force-spinning, orpreferably through electrospinning, of a fibroin solution.

The starting solution is prepared by dissolving fibroin in a solventselected from formic acid, 1,1,1,3,3,3-hexafluoro-2-propanol,trifluoroacetic acid, or ionic liquids such as1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazoliumchloride, 1-ethyl-3-methylimidazolium acetate,1-ethyl-3-methylimidazolium glycine, 1-allyl-3-methylimidazoliumchloride, 1-butyl-2,3-dimethylimidazolium chloride,1-butyl-3-methylimidazolium bromide and mixtures among these solventsand/or with water; the preferred ionic liquids for the purposes of theinvention are 1-ethyl-3-methylimidazolium chloride,1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazoliumacetate and 1-ethyl-3-methylimidazolium glycine.

The starting solution has a fibroin concentration of between 1% w/v and30% w/v, preferably between 6% w/v and 10% w/v, in formic acid; oralternatively, a fibroin concentration of between 5% w/v and 40% w/v,preferably between 10% w/v and 30% w/v, in the other solvents indicated;the weight/volume (% w/v) percentage concentration indicates the gramsof fibroin dissolved in 100 mL of solution.

The preferred method of production of the nanofibrous fibroin part iselectrospinning, the general execution methods of which are known to theman skilled in the art: in order to obtain a nanofibrous fibroinsuitable for the purposes of the invention, the solution is electrospunwith a potential difference between the nozzle of the spinneret and thecollector between 5 kV and 100 kV, preferably between 15 kV and 35 kV,with a distance between said nozzle and collector between 5 cm and 60cm, preferably between 10 cm and 20 cm. The nozzle of the spinneret canhave diameters of between 0.01 mm and 10 mm, preferably between 0.1 mmand 1 mm.

Both in the case of force-spinning and of electrospinning, the startingsolution may be added with bioactive agents including growth factors,drugs, cells, antibiotics, antiviral agents, enzymes, vitamins, etc.,which are thus integrated in the medical device and can then be releasedfrom the same at the implantation site, in order to promote theregenerative processes of the body area said device is intended for. Thenanofibrous fibroin can be added with bioactive agents including growthfactors, drugs, cells, antibiotics, antiviral agents, enzymes, vitamins,etc., for example by means of chemical and/or enzymatic reactions. Thisaddition may be made during any of steps c) to g), or later on.

The previous steps have been named a) and b) only for the purposes ofclarity of illustration, but this does not imply a temporal order ofexecution; nano and microfibrous parts are produced separately, and thetwo steps may be carried out in any sequence.

After their preparation, in step c) of the process, the parts ofmicrofibrous and nanofibrous fibroin are treated with a solvent or asolution containing additional fibroin. In this step, a surface fractionof the fibers passes to the gel phase, forming a film around the presentfibers. The treatment with said solvent or solution can take place onsaid separate parts, or after having put them in contact with oneanother.

In the case of treatment with solvent alone, this is selected from: anionic liquid that can be 1-ethyl-3-methylimidazolium chloride,1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazoliumacetate, 1-ethyl-3-methylimidazolium glycine,1-allyl-3-methylimidazolium chloride, 1-butyl-2,3-dimethylimidazoliumchloride, 1-butyl-3-methylimidazolium bromide or mixtures thereof, pureor in mixture with water; formic acid; trifluoroacetic acid;1,1,1,3,3,3-hexafluoro-2-propanol; hexafluoroacetone; calciumchloride-ethanol-water mixtures; calcium nitrate-methanol-watermixtures; N-methylmorpholine N-oxide; or aqueous solutions of lithiumsalts (lithium bromide, lithium thiocyanate). The preferred solvents forthis treatment are 1-ethyl-3-methylimidazolium chloride,1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazoliumacetate, 1-ethyl-3-methylimidazolium acetate or1-ethyl-3-methylimidazolium glycine, pure or mixtures thereof with waterwith a water content of between 5% w/w and 50% w/w and preferablybetween 10% w/w and 25% w/w; another preferred solvent for the purposesof the invention is formic acid.

The exposure to the solvent of the nanofibrous and microfibrous fibroinparts (either separate or already in contact with one another) can takeplace according to different methods, such as for example:

-   -   immersion in the solvent, for a time between 1 second and 240        minutes, preferably between 30 seconds and 30 minutes;    -   deposition of the solvent by pouring, coating, atomizing,        electrospray or electrospinning, in an amount between 0.001        mL/cm² and 0.5 mL/cm², preferably between 0.01 mL/cm² and 0.1        mL/cm²; these amounts are referred to the apparent surface of        the parts, i.e. those deduced from the simple multiplication of        the length and width of the parts themselves (the contribution        of the height to the surface of the part is in general        negligible), and not to the overall surface of the individual        fibers;    -   exposure to the vapors of the solvent, for a time between 1        second and 120 minutes, preferably between 30 seconds and 30        minutes.

The temperature at which the contact between the microfibers and thesolvent is made is variable between 40 and 80° C., preferably between 50and 70° C.; it is also possible to operate at temperatures lower than40° C., but in this case the process execution time becomes very longand not suitable for an industrial production. For the nanofibers, thetemperature at which contact with the solvent is made is variablebetween room temperature and 70° C., preferably between 40 and 60° C.

In the case of separate treatment of the two parts prior to theircoupling, it is possible to treat one of the two with solvent alone, andthe other with a solution of fibroin in a solvent (not necessarily thesame as that of the treatment with solvent alone).

It is also possible to treat one or both parts in succession with thesolvent alone to cause an initial gelling of the fibers, and then with asolution of fibroin to impart an additional aliquot of dissolved polymerand facilitate the subsequent coupling of the parts.

For an optimal execution of the process of the invention, the contacttime between fibers and solvent should be reduced, in the rangesmentioned above, with increasing temperature and with decreasingdimensions of the yarns or dimensions of the parts to be treated. Forexample, for the same thickness of the microfibers, suitable contacttimes with the solvent will be between about 30 seconds and 3 minutes attemperatures between 70 and 80° C., and between about 15 minutes and onehour for temperatures between 40 and 50° C. As regards the nanofibers,these contact times range between 30 seconds and one minute at 70° C.,and between about 5 and 30 minutes at temperatures between 40 and 50° C.

The contact times, with the same temperature, also vary depending on theapparent density of the part, i.e. the amount of fibers per unit ofvolume of the same, especially in the case of parts made of microfibers;still remaining in the general ranges mentioned above, the suitablecontact time for a fabric decreases, for example passing from crêpe totwill, from twill to organdie and from organdie to non-woven fabric.

Taking into account these general guidelines, the man skilled in the artis able to choose the optimal operating conditions suitable forobtaining the effective coupling of the available microfiber andnanofiber parts.

In the case of treatment with a fibroin solution, the solution isproduced with the same solvents mentioned above for treatment with thesolvent alone. Preferably, the solution is based on1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazoliumchloride, 1-ethyl-3-methylimidazolium acetate or1-ethyl-3-methylimidazolium glycine, pure or mixed with water (watercontent between 5% w/w and 50% w/w, preferably between 10% w/w and 25%w/w), with a concentration of fibroin between 5% w/v and 40% w/v,preferably between 10% w/v and 30% w/v; or based on formic acid with aconcentration of fibroin between 1% w/v and 30% w/v, preferably between6% w/v and 10% w/v.

The contact between the fibroin solution and the nanofibrous andmicrofibrous fibroin parts can occur through immersion in solvent ordeposition of the same on said parts (already in contact with oneanother or still separate), with the same methods, timing and quantityamounts reported above in case of treatment with solvent alone.

If said parts were not already in contact with one another before saidtreatments with solvent or solution, these are put in contact with oneanother in step c′), obtaining a micro/nanofibrous hybrid structure.

In the case of planar geometry devices, the nanofibrous and microfibrousparts are stacked (placed) on top of one another according to desirednumber and sequences. In the case of tubular medical devices, thenanofibrous and microfibrous parts are “fitted” on top of one anotheraccording to desired number and sequences. In the case ofthree-dimensional hybrid structures other than tubular, the nanofibrousand microfibrous parts are made to adhere to one another according todesired number and sequences.

In steps c) and c′) it is possible to add the hybrid structure beingformed with bioactive agents, including growth factors, drugs, cells,antibiotics, antiviral agents, enzymes, vitamins, etc., dispersing suchbioactive agents in the solvents and/or solutions used. In this way, thebioactive agents come into contact with the component parts of thehybrid structure and can be incorporated therein and therefore in theimplantable medical devices in which this is used.

In step d) of the process, the hybrid structure thus obtained is thensubjected to heat treatment to strengthen the coupling of the two ormore fibroin parts, of which at least one of nanofibrous fibroin and atleast one of microfibrous fibroin. This treatment is carried out on thehybrid structure still impregnated with solvent, at a temperaturebetween 10° C. and 150° C.; preferably, the treatment temperature isbetween 80° C. and 120° C. when an ionic liquid is used in the previousstep, and between 20° C. and 50° C. when formic acid or other solventsare used in the previous step. The duration of treatment is between 1minute and 24 hours.

Preferably, during step d), the set of parts to be coupled is subjectedto compression, typically at values between 0.5 and 5 kg/cm², preferablybetween 0.1 and 1 kg/cm², to increase the efficiency of the distributionprocess onto both types of fibers of the gelified fibroin formed in stepc).

This operation can be carried out for example by means of an apparatussuch as that shown in FIG. 1. The apparatus includes a heating element10 at the bottom, upon which a plate 11 rests; in its upper part, theapparatus includes a second plate 12. The two plates must be made ofrigid, thermally conductive and non-stick material. The heating elementmust allow a fine control of the temperature, in the range of treatmenttemperatures mentioned above, with an accuracy of ±0.5° C., better if±0.1° C., in order to allow the management of the gelling process withthe necessary accuracy. According to the preferred operating methoddescribed above, a light pressure (shown in the figure by the arrowdirected downwards and by the indication “P”) is applied on the topplate during the heating step using a weight and/or a closing systemthat brings the two plates close to each other so as not to leave anyempty space between the fibroin parts, nor between said parts and theplates. Finally, the device is preferably locked in a heat-insulatingchamber, accessible from the outside, in order to prevent heat lossesthat might impair the effectiveness of the gelling process. The optimalarrangement of the two parts of fibroin to be coupled is as shown in thefigure, with the microfibrous part 14, less sensitive to the effects ofheat combined with the ionic liquid, in contact with plate 11 adjacentto the heating element, while the nanofibrous part 13 is in contact withthe top plate 12. This configuration (microfibrous part closer to theheating element and nanofibrous part farther therefrom) is preferablealso in the case of hierarchically superior hybrid structures obtainedaccording to steps f) and g) of the process, when allowed by thestacking order of the parts.

In the case of tubular parts, the coupling is carried out for examplewith an apparatus consisting of a cylinder that may be solid or hollow,made of rigid, thermally conductive and non-stick material and with adiameter slightly greater than the diameter of the parts to be coupled(with a difference in diameter preferably between 0.05 mm and 3 mm). Thespindle must be connected to a system suitable for adjusting thetemperature thereof between 10° C. and 150° C. and it may be containedin a heat-insulating chamber; this chamber may be thermostaticallycontrolled and allow temperature control in the same range. In thisoperating mode, one of the parts is fitted on the spindle and then theother part is fit onto the first one, in both cases taking advantage ofthe elasticity of the fibroin parts. The difference in size between thespindle and the parts generates a radial pressure of the outermost partson the innermost parts, promoting the adhesion and the coupling of thesame parts. The preferred configuration in this operating mode isdetermined by the final objective of the produced device; therefore, byway of example, for the manufacture of medical devices for therepair/regeneration of blood vessels, the nanofibrous part will bemounted in contact with the spindle while the microfibrous part will befitted on the nanofibrous part.

In step e) of the process, the solvent is removed by washing with wateror a water-alcohol mixture, or by evaporation. Washing is carried out ata temperature between 10° C. and 100° C., preferably between 20° C. and50° C., for a time between 2 minutes and 180 minutes, preferably between10 minutes and 60 minutes; the preferred alcohols in case of use ofwater-alcohol mixtures are methanol and ethanol, with waterconcentrations between 5% v/v and 50% v/v and preferably between 10% v/vand 30% v/v. If the solvent is removed by evaporation, this is carriedout at a temperature between 10° C. and 100° C. possibly under vacuum,preferably between 15° C. and 55° C., for a time between 10 minutes and168 hours and preferably between 30 minutes and 72 hours.

Steps c) and d) of the process lead to a partial surfacesolubilization/gelification of the fibrous material making up theadjacent parts. The next step e) of solvent removal causes thecoagulation of the gelified fibroin fraction; the passage of fibroinfrom the gel state to the solid (coagulated) state that takes place inthe contact areas between the structures leads to the formation of“junctions”, union and fusion points between microfibers and nanofibers.This forms a hybrid structure consisting of microfibers and nanofiberswelded together and constituting a single part.

Once a micro/nanofibrous hybrid structure has been produced as describedabove, this can be coupled to another or to several similar structuresto form hierarchically organized complex structures, by carrying out theoptional steps f) and g) mentioned above.

The second aspect of the invention relates to the implantable medicaldevices that use the hybrid structures obtained with the processdescribed thus far.

The implantable medical devices of the invention are mainly used asscaffolds for repairing human and animal tissues and organs withregenerative medicine approach. Among the target tissues and organs wemay mention, among the others, tissues of the peripheral nervous system(peripheral nerves), vascular system (veins, arteries, arteriovenousfistulas for vascular access), cardiovascular system (coronary arteriesand the heart muscle), central nervous system (spinal cord), skin andits layers, containment and protection tissues of internal organs (duramater, pericardium, pleura, peritoneum, . . . ), tissues of themusculoskeletal system (tendons, ligaments, muscles) and devices for thecontainment of hernias and prolapses.

The shape and size of the device depend on the target tissue or organ;below are listed the rough shapes and sizes of devices intended for someof the purposes mentioned above and achievable using the hybridstructures of the invention, but other possible uses (and the shapes andsizes of relevant devices) will be apparent to the man skilled in theart:

-   -   tubular devices with inner diameter of between 2 mm and 8 mm and        wall thickness of between 0.2 mm and 4 mm in the case of        peripheral blood vessels;    -   tubular devices with inner diameter of between 1 mm and 6 mm and        wall thickness of between 0.2 mm and 1 mm in the case of        peripheral nerves;    -   solid cylindrical devices with outer diameter of between 2 mm        and 15 mm in the case of tendons and ligaments;    -   planar devices with thickness of between 0.1 mm and 5 mm in the        case of the skin and its layers;    -   planar devices with thickness of between 0.05 mm and 2 mm in the        case of containment and protection tissues of internal organs        such as dura mater, pericardium, pleura, peritoneum;    -   planar devices with thickness of between 0.05 mm and 2 mm in the        case of devices for the containment of hernias and prolapses.

The invention will be further illustrated by the following examples.

Example 1

This example describes the production of microfibrous fibroin filamentsand of parts (fibers, fabrics) obtained from these filaments.

Cocoons of B. mori were subjected to spinning to produce a raw silkyarn. After doubling and twisting, the yarn was scoured with water underpressure at 120° C. for 30 min to remove the sericin. For the productionof fabrics, the raw yarn was first woven in the desired weaves and thefabric thus obtained was then scoured at 95-98° C. for 1 hour, in thepresence of surfactants to remove the sericin. To produce the non-wovenfabric, the cocoons were cut and macerated to remove the sericin. Theshort fiber silk thus obtained (called “shappe”) was subjected tocarding. The veil of card was consolidated into non-woven by needling.

The following was produced with the thus obtained filaments:

-   -   a scoured silk yarn (3 yarn weft; count 17.1×3 den);    -   a scoured silk fabric having the following features: weave:        crêpe; no. yarns/cm in warp: 58 (count: 15.3×3 den); no.        yarns/cm in weft: 39 (count: 15.3×3 den); mass per unit area: 55        g/m²; thickness 0.12 mm;    -   a scoured silk fabric having the following features: weave:        organdie; no. yarns/cm in warp: 53 (count: 20.7 den); no.        yarns/cm in weft: 39 (count: 23.4 den); mass per unit area: 30        g/m²; thickness 0.09 mm;    -   a scoured silk fabric having the following features: weave:        twill; no. yarns/cm in warp: 55 (count: 15.3×3 den); no.        yarns/cm in weft: 43 (count: 15.3×4 den); mass per unit area: 60        g/m²; thickness 0.09 mm;    -   a non-woven fabric (TNT) of scoured silk, having the following        features: fiber length: 20-27 mm; mass per unit area: 33 g/m².

These samples are used for the tests of the following examples.

Example 2

This example describes the production of nanofibrous fibroin parts.

Cocoons of B. mori were scoured with distilled water in autoclave at120° C. for 30 min, to remove the sericin.

After thoroughly rinsing and drying at room temperature, 1 g of fibroinmicrofibers was dissolved in 10 mL of a saturated solution of LiBr(about 9.3 M) for 3 hours at 60° C. After dilution with an equal volumeof distilled water, the fibroin solution was dialyzed for 3 days againstdistilled water to remove the salt. The resulting fibroin solution wasdiluted to 67 mL with water, resulting in a 1.5% w/v aqueous solution offibroin. The solution, divided into 15 mL aliquots, is poured into moldswith a diameter of 5 cm and allowed to evaporate at room temperature,obtaining fibroin films having an average thickness of 50 μm.

Just before the electrospinning process, 2 g of film are dissolved in 25mL of formic acid at room temperature, obtaining a solution with apolymer concentration equal to 8% w/v.

For the production of nanofibrous fibroin parts, the fibroin solution informic acid is loaded in a polypropylene syringe attached to a syringepump (Graseby Medical, MS 2000) with a PTFE capillary tube. Theelectrospinning system consists of two high voltage power supplies(F.u.G. Elektronik GmbH, HCN 35-12500) capable of generating up to 25kV. The positive pole is connected to the spinneret, consisting of asteel capillary tube with an inner diameter of 0.5 mm, able to move indirection transversal to the collector. The negative pole is connectedto the collector, consisting of a rotating cylinder of 20 cm×8 cm (I×d);nanofibroin parts are obtained in this way in the form of hollowcylindrical bodies, which are then cut lengthwise and laid out to formgenerally flat parts. Several samples of electrospun fibroin parts areproduced using the following experimental parameters: concentration offibroin=8% by weight; voltage=24 kV; flow=3 mL/h; spinneret/collectordistance=10 cm; harvest time=6 hours. At the end of electrospinning, thefibroin parts are detached from the collector, treated with awater-alcohol solution for 30 min at room temperature and air-dried.These parts have an average thickness of 50 μm.

Example 3

The test described in this Example is intended to determine the amountof ionic liquid which may be retained in different parts of nano- ormicrofibrous fibroin.

The properties of the four microfibrous fibroin fabrics mentioned inExample 1 (organdie, crêpe, twill and non-woven fabric) and of a part ofnanofibrous fibroin of Example 2 are evaluated.

The ionic liquid used for the test is 1-ethyl-3-methylimidazoliumacetate.

The test is carried out according to two impregnation methods, byimmersion of the samples in the liquid followed by draining dripping bygravity, and by surface deposition with a brush. In both cases, theamount of liquid retained immediately after the impregnation and aftersqueezing is evaluated, measured as a percentage by weight with respectto the sample weight; squeezing is carried out by compressing thesamples obtained by immersion with a force of 0.5 kg/cm² for 60 minutes,and compressing the samples obtained by surface deposition with a forceof 0.1 kg/cm² for 2 minutes. The obtained results are shown in Table 1.

TABLE 1 Amount of liquid retained by the sample (% by weight) ImmersionSurface deposition Without After Without After squeezing squeezingsqueezing squeezing Nanofiber 503 ± 124 55 ± 12 81 ± 8 11 ± 1  Organdie394 ± 16  18 ± 2  131 ± 4  16 ± 4  Crêpe 280 ± 8  6 ± 3 154 ± 15 40 ± 4 Twill 325 ± 6  6 ± 2 155 ± 23 35 ± 10 TNT 4850 ± 750  22 ± 2  n/a n/a

Example 4

Coupling of nano- and microfibrous parts according to the invention.

A sample of organdie fabric and one of crêpe fabric of Example 1, and asample of nanofibrous part of Example 2, having a size of 3×5 cm, aretreated with ionic liquid with surface coating and squeezing as inExample 3.

An organdie/nanofibrous part hybrid structure and a crepe/nanofibrouspart hybrid structure are then produced with the materials thusimpregnated, introducing the coupled materials in the apparatusschematized in FIG. 1.

Each pair of materials is introduced in said apparatus with themicrofiber layer (organdie or crêpe) at the bottom, in direct contactwith the heating plate; a slight pressure (0.1 kg/cm²) is applied to thetop plate. The apparatus is placed in a thermostatic chamber to preventheat losses, and the temperature of the bottom plate is raised to 55° C.for 5 minutes. At the end of this period, the apparatus is removed fromthe thermostatic chamber and allowed to cool down to room temperature(in about 10 minutes), after which a mixture at a concentration of 80%w/w of ethyl alcohol in water is injected between the two plates with asyringe.

The plates are then opened and the hybrid structure is transferred in abath of the same water-alcohol mixture to remove all traces of residualionic liquid; the hybrid structure is left in this bath for 24 hours.

At the end of this period, the hybrid structure is rinsed in distilledwater to remove the alcohol and placed between several layers of papertowels which are changed periodically until complete drying of thestructure (taking about 12 hours).

Example 5

Chemical characterization of nano- and microfibrous hybrid structures ofthe invention.

The amino acid composition of separate fibroin microfibrous andnanofibrous parts and of the hybrid structure obtained in Example 4 wasevaluated.

About 25 mg of material for each of the three samples were hydrolyzedwith HCl 6N, at 105° C., for 24 hours under vacuum. The hydrolysatesolutions thus obtained were analyzed with an automatic ion exchangeamino acid analyzer. The results of the analysis are shown in Table 2.

TABLE 2 Amino acids (mol %) Hybrid Micro part Nano part structureAspartic acid 1.91 1.66 1.55 Threonine 1.22 0.84 1.49 Serine 11.10 10.5711.10 Glutamic acid 1.25 1.48 1.34 Proline 0.68 0.84 0.82 Glycine 43.8244.88 44.47 Alanine 29.34 29.54 28.93 Cystine — — — Valine 2.28 2.312.10 Methionine — — — Isoleucine 1.08 0.94 1.32 Leucine 0.65 0.45 0.68Tyrosine 4.80 4.73 4.54 Phenylalanine 0.37 0.56 0.46 Lysine 0.53 0.310.44 Histidine 0.30 0.23 0.25 Arginine 0.66 0.67 0.50 Total 100.00100.00 100.00

Example 6 (Comparative)

Coupling of nano- and microfibrous parts according to the prior art.

For comparison purposes, four samples consisting of coupled fibroinnanofibers and microfibers are produced following the procedure ofdocument CN 101879330 A.

Using the same starting materials of Example 4, two hybrid structuresare produced in accordance with the following procedure:

the nanofiber part and the microfiber fabric (organdie or crêpe) arebrought into contact and impregnated with an aqueous solution of fibroinat 4% by weight;

the resulting coupled system is treated at 60° C. for 30 minutes andsubsequently immersed in a water-alcohol solution at 80% w/w of methanolfor 15 minutes;

the two coupled systems are then subjected to drying at roomtemperature, under controlled conditions of temperature and humidity(20° C., 65% relative humidity).

The two samples of hybrid structure thus obtained are hereinafterreferred to as “SF Film”.

Example 7 (Comparative)

Coupling of nano- and microfibrous parts according to the prior art.

The procedure of Example 6 is repeated, the only difference being thatafter bringing the fibroin parts into contact and impregnating them withthe aqueous fibroin solution at 4% by weight, the system is consolidatedby freezing at −20° C. and subsequent freeze-drying.

The two samples of hybrid structure thus obtained are hereinafterreferred to as “SF Gel”.

Example 8

Morphological characterizations of nano- and microfibrous hybridstructures of the invention and of the prior art.

The organdie/nanofibrous part hybrid structure produced in Example 4 wasobserved with scanning electron microscope (SEM, mod. MIRA 3, Tescan).For comparison, samples of organdie fabric and of nanofibrous part werealso observed before coupling. The organdie fabric was chosen as theopen arrangement of the warp and weft yarns leaves some gaps throughwhich it is possible to characterize the surface of the nanofibrous parton the side adjacent to the microfibrous part (coupling side).

For this purpose, 0.5×0.5 mm samples were taken from the hybridstructure, positioned on aluminum sample-holders for SEM with adouble-sided adhesive tape, and coated with gold-palladium bysputtering. Both sides exposed to the air were examined, that of themicrofibers and that of the nanofibers.

Photomicrographs of the samples are shown in FIG. 2.

FIG. 2-A and FIG. 2-B show the microfibrous organdie fabric and thenanofibrous part, respectively, before coupling; the latter has thetypical features of a substrate obtained by electrospinning, withfibroin nanofibers having an average diameter of 500-600 nm, laidirregularly (as non-woven fabric) and with a very fine porosity. Aftercoupling, the microfibrous part (FIG. 2-C) displays a slight flatteningof the component yarns, probably due to the pressure exerted in thevarious steps of the coupling process; the warp and weft yarns howeverstill retain their original structure and the individual microfibers ofwhich they are made are still well visible.

Among the pores of the fabric (FIGS. 2-D and 2-E), the surface of thecoupled nanofibrous part may be seen, which retains the typicalroughness visible at low magnification; in some areas (FIG. 2-E), partlygelified areas may be seen with more evidence which connect microfibersand nanofibers and keep them in close contact.

FIG. 2-F shows the surface of the nanofibrous part exposed to air aftercoupling. Although there are fusion areas of the nanofibers, the typicalmorphology observed for the untreated native part is retained.

FIGS. 3-A and 3-F (especially FIG. 3-F) show the presence of a thinlayer of gel which coats both the microfibers and the nanofibers,connecting them with interfibrous connections. The gel layer isextremely thin and superficial, showing through the surface of thesingle microfibers and also that of the nanofibers, whose morphology isonly slightly deformed on the surface while it is substantially retainedin the remaining part of the material.

Finally, FIG. 3-B shows a peripheral area of the hybrid structure, alongthe edge of the cutting with which the sample subjected to SEMobservation was taken. This image shows that the coupling process of theinvention is effective and that the two coupled parts do not separateeven if subjected to deformation and compression, as usually happens inthe area subject to cutting with scissors or scalpel.

For comparison, a sample of the prior art is examined under SEM,produced according to the procedure of comparative Example 6 (organdie“SF Film” sample). The images of this sample are shown in FIGS. 4-A and4-B, and they show the presence of a film on the surface of themicrofibers, crushed and adhering only to the latter and unable to serveas an effective adhesive with the nanofibrous part.

Example 9

Chemical-physical characterization of hybrid structures of theinvention.

The organdie/nanofibrous hybrid structure produced in Example 4 isfurther characterized by Fourier transform infrared (FTIR) spectroscopyin order to verify whether the coupling process causes changes in thephysical-chemical, structural and conformational properties of themicro- and nanofibrous components.

A NEXUS Thermo Nicolet spectrometer in ATR (Attenuated TotalReflectance) mode was used, with Smart Performer accessory equipped witha SeZn crystal cell. FTIR spectra were recorded in the wavenumber range4000-700 cm⁻¹, accumulating 64 scans at a resolution of 4 cm⁻¹. Eachspectrum is the average of three measurements (FIG. 5).

The spectral region 1900-700 cm⁻¹ represents the fibroin fingerprintfrom the point of view of the composition and structure of the polymer.The most significant conformationally sensitive bands are known as AmideI (1615-1690 cm⁻¹), Amide II (1509 cm⁻¹), and Amide III (1230-1260cm⁻¹), derived from a multiplicity of vibrational modes of the peptidebond. Amide I is mainly due to stretching vibrations of the CO bond,with a contribution of the CN bond; Amide II is due to the bending ofthe NH bond (predominant) with the contribution of the stretching of theCN bond; Amide III is due to NH bending and CN stretching vibrations.

FIG. 5-A shows, overlapped, the spectrum of microfibers before treatment(curve (a)) and of the hybrid structure of invention (curve (b));similarly, FIG. 5-B shows, overlapped, the spectrum of nanofibers beforetreatment (curve (a)) and of the hybrid structure of invention (curve(b)).

Based on the position and intensity of the bands of Amide I, II and IIIin the spectra, it can be deduced that both the microfibers and thenanofibers, before the coupling treatment, have the typical β-sheetmolecular conformation, characteristic of native (microfibers) orregenerated (films, nanofibers, etc.) crystalline fibroin materials.

The spectral profiles after coupling are exactly superimposed to thoseof the respective untreated samples, indicating that the structuralfeatures of the material are retained.

The two components of the band of Amide III were used to calculate thecrystallinity index of the materials before and after the couplingprocess. The crystallinity index is obtained from the ratio between theintensity of the band at 1260 cm⁻¹ and that of the band at 1230 cm⁻¹(C_(I)=I₁₂₆₀/I₁₂₃₀). For microfibers, this index remains essentiallyunchanged after coupling, changing from 0.52 to 0.51, while fornanofibers it decreases by about 8%, from 0.60 to 0.55. This behavior isconsistent with the transformation of a fraction of the nanofibrous partthat during the gelification process and the subsequent coagulationtakes a less orderly structure than the pre-existing one, changing intoa transition phase with adhesive properties, as shown by thephotomicrographs in Example 8.

Example 10

Structural characterization of hybrid structures of the invention.

The organdie/nanofibrous part hybrid structure produced in Example 4 isfurther characterized by differential scanning calorimetry (DSC).

A calorimeter 200 Q TA Instruments is used, recording the curves fromroom temperature to 500° C. with heating rate of 10° C./min undernitrogen flow; each sample, weighing about 5 mg, was introduced inaluminum crucible and analyzed in duplicate. The test results are shownin FIG. 6, which shows the thermograms for microfibers alone (curve(a)), for nanofibers alone (curve (b)) and for the hybrid structure ofthe invention (curve (c)).

As can be seen, all curves show a first endotherm at a T below 100° C.which can be ascribed to the evaporation of the moisture containedwithin the material.

In the case of microfibers, a second, very intense endotherm follows,with peak at 313° C., attributed to the thermal degradation of fibroinin the form of crystalline and oriented fiber with β-sheet conformation.

The thermogram of nanofibers before treatment (curve (b)) has a similarprofile, in which however the second endotherm is at lower temperature(282° C.), indicating a much lower orientation degree of the crystallinephase and much more irregular crystal size than in the case ofmicrofibers.

The thermal diagram of the hybrid structure sample (curve (c)) shows thecharacteristic transitions of the two component parts: the degradationpeak of nanofibers at 282° C. remains unchanged, while that ofmicrofibers moves to 308° C., possibly due to intermolecularinteractions in the areas of very close mutual contact present in thecoupled materials of the invention.

Example 11

Mechanical characterization of hybrid structures of the invention and ofthe prior art.

Tensile tests are carried out on a sample of Example 4, consisting ofthe coupling of a nanofibrous part (50 μm thickness) and of an organdieweave fabric as microfibrous component (90 μm thickness); forcomparison, also the properties of strips of the separate nanofibrouspart and of the microfibrous fabric were measured.

The thickness of the samples before and after coupling was measuredaccording to the standard UNI EN ISO 5084:1998 method. The obtainedvalues were used to calculate the mechanical parameters of stress andmodulus. The mechanical properties were measured on strips of parts assuch and of hybrid structure, having a size of 20×10 mm (length×width),using an Instron dynamometer mod. 4501, at 10 mm gauge length, and 10mm/min crossbar rate. The measurements were carried out in standardatmosphere at 20° C. and 65% relative humidity. The stress, deformationand modulus values were calculated from the load-elongation curves andthey represent the average of ten measures for each sample.

The obtained results are shown in the graphs in FIG. 7 and summarized inTable 3.

The load-elongation curves of the microfibrous part (FIG. 7-A) arecharacterized by an initial step of stretching of the yarns that make upthe textile structure. Once stretched, an additional elongation occursascribable to the inherent characteristics of elasticity of the yarn,which oppose an increasing resistance to elongation itself, as shown bythe gradual increase of the load values. Finally, breaking occurs afteran elongation of about 50%. The salient features of this part are a hightoughness, an equally high elongation and a relatively low initialmodulus.

Conversely, the nanofibrous part (FIG. 7-B), has a diametricallyopposite mechanical tensile response: low toughness and elongationvalues, very high initial modulus.

The hybrid structure of the invention (FIG. 7-C) displays a mechanicalbehavior that is not simply the result of the sum of the individualcomponents of the system, but rather, it displays deviations fromadditivity which account for a very strict and specific interactionbetween the two component parts. In fact, the hybrid structure ischaracterized by a very high initial resistance to the applied load.This resistance is attributed to the close interaction between the twoparts at their interface. As the load increases, the load-elongationcurves become jagged, which is attributed to the sequential rupture ofthe contact points between the microfibrous and nanofibrous parts. Thisphase extends to elongation values of 20-25%, significantly higher thanthose of the nanofibrous part as such, which does not stretch more than6-7%. The further increase in load brings out the contribution of themicrofibrous part which then breaks for elongations between 45% and 50%,a value very similar to that of the part as such.

The mechanical values measured during the tests, also including stressand modulus, are shown in FIG. 7-D and summarized in Table 3.

TABLE 3 Hybrid Micro/ Microfibers Nanofibers Nanofibers Stress (MPa)63.7 ± 0.5 20.8 ± 5.0  41.9 ± 1.6 Elongation (%) 48.0 ± 1.3 6.4 ± 1.544.7 ± 3.3 Modulus (MPa) 14.9 ± 1.1 82.2 ± 7.0  56.5 ± 7.2

Example 12 (Comparative)

For comparison, the test of Example 11 is repeated on a sample of theprior art (“SF Gel” sample with organdie fabric, produced as describedin Example 7).

A sheet of porous fibroin alone was also produced, following the sameprocedure and pouring the aqueous solution, before freezing andfreeze-drying, into a mold.

20×10 mm strips of these samples were subjected to the same test as theprevious Example, in identical conditions.

The results are shown in FIG. 8 for the porous sheet (A) and for themicro/nano hybrid structure (B), respectively.

The porous sheet (FIG. 8-A) has very poor mechanical properties: theaverage value of load strength is 1.1±0.2 N, about 10 times lower than ananofibrous part (11.2±1.8 N: see FIG. 7-B); the variability of thecurve shape is due to the heterogeneity of the texture of porousmaterials of silk fibroin obtained with the method described above.

The load-elongation curves of the micro/nano hybrid structure (B)coupled using porous fibroin (FIG. 8-B) are characterized by thepresence of two peaks, one at low and the other at high deformationvalues. The peak at low deformation corresponds to the breaking of thenanofibrous component, as indicated by the load and elongation at breakvalues (15±2 N and 5.5±0.2%, respectively).

The peak at high deformation corresponds to the breaking of themicrofibrous substrate (load: 40±3 N; elongation at break: 29±3%).

Example 13

Measurement of the adhesion strength of hybrid structures of theinvention and of the prior art.

Two samples of the invention of Example 4, prepared from organdie andcrêpe fabrics, and two samples (organdie and crêpe) for each of the “SFFilm” and “SF Gel” materials of the prior art, produced as described inExamples 6 and 7, respectively, are subjected to mechanical testsdesigned to measure the adhesion strength between the two micro andnanofibrous components of the hybrid structures. The tests were carriedout using an Instron dynamometer mod. 4501, according to the standardUNI EN ISO 13937-2:2000 method.

In particular, 10×40 mm rectangular strips are taken from each of thesamples. At one end of the sample, the two flaps, one corresponding tothe nanofibrous part and one to the microfibrous part, were delicatelyseparated by a stretch of about 10 mm; these flaps were locked into thegrips of the dynamometer, as shown in FIG. 9. The top (movable) grip wasthen actuated, moving it away from the bottom (fixed) one at 2 mm/mincrossbar rate, and the force required (measured in cN) to cause theseparation of the microfibrous (lower) part from the nanofibrous (upper)part was recorded continuously for a stretch of at least 20-30 mm. Atleast 5 tests were carried out for each sample and the single resultswere averaged. Typical “peel load/stroke” curves are shown in FIG. 10-A.The results obtained from the processing of experimental data are shownin Table 4 and in the bar chart in FIG. 10-B.

TABLE 4 Separation force (cN) Invention “SF Film” “SF Gel” Mean MaximumMean Maximum Mean Maximum Crepe Microfiber 53 ± 11 72 ± 12 29 ± 6 58 ±9  11 ± 5 18 ± 5 Organdie Microfibers 21 ± 9  31 ± 11 10 ± 5 24 ± 10  5± 2  9 ± 2

Example 14

In vitro cytotoxicity and genotoxicity studies.

In view of the application for the production of scaffolds forimplantation in the human and animal body, the in vitro biologicalproperties of the composite materials of the invention were evaluated.

The tests were conducted with two human cell models, human fibroblasts(MGM18004E) and human endothelial cells (HUVEC).

Human fibroblasts were cultured in DMEM culture medium with a highglucose content (Gibco) containing 20% bovine fetal serum inactivated byheat treatment (Gibco), 200 mM L-glutamine (Euroclone), penicillin andstreptomycin (Euroclone).

Human endothelial cells were cultured in EBM-2 (basal medium forendothelial cells 2, Lonza) culture medium, containing penicillin andstreptomycin (Euroclone).

Analytical tests were designed to assess the degree of cellproliferation and DNA damage as markers of potential cytotoxicity andgenotoxicity of the biomaterial.

The Alamar Blue® test measures the metabolic activity of cells, which isdirectly linked to cell proliferation. The cells were seeded in 96-wellplates at an initial density of 6000 cells/cm². Culture medium alone andcells alone were used as blanks. The tests were carried out in technicaltriplicate and biological duplicate. Cells were cultured for 24, 72 and120 hours in an incubator at 37° C., in the presence of 5% CO₂. Theculture medium was changed on day 3. At the end of the incubationperiod, a fixed volume of Alamar Blue® (10% of total volume) was addedto the well. After a further incubation period of 18 hours, the mediumwas transferred to another plate and the absorbance values at 570 nm and600 nm were recorded with a multidisc reader (Biotech). Results wereexpressed as percentage difference between samples with cells alone andsamples into contact with the biomaterial of the invention.

The DNA damage test evaluates the possible genotoxicity of thebiomaterial by detecting the presence of phosphorylated Ser₁₃₉ in theH2AX histone. Phosphorylation is induced by the presence of ruptures inthe DNA double strand by immunofluorescence. The cells were seeded at aninitial density of 6000 cells/cm² for 24 hours, 3000 cells/cm² for 120hours, and 1500 cells/cm² for 120 hours. The culture medium was changedon day 3. Cells treated with 200 mm H₂O₂ for 16 hours were used aspositive control. At the end of the experiment, cells were fixed with 4%paraformaldehyde (Sigma-Aldrich), followed by permeabilization withphosphate saline buffer solution (PBS) containing 0.1% BSA (Bovine SerumAlbumin) and 0.25% Triton X-100. Non-specific reaction sites wereblocked by incubation with blocking buffer (0.1% BSA in PBS).Subsequently, an anti-γH2AX antibody was incubated for 1 hour, andrevealed through a secondary goat antibody Alexa Fluor® 555 anti-mouseIgG. The cell nuclei were marked with Hoechest 33342. The plates wereexamined with a Leica DMI4000B fluorescence microscope (LeicaMicrosystems) at 20×. The average number of positive cells to DNA damagewas determined by observing 3-5 independent fields for each biologicalrepetition and for each experimental condition.

FIG. 11 shows the cellular growth curves, expressed as a percentage ofthe control growth. At 24 hours, human fibroblasts show a degree ofproliferation similar to that of cells seeded into the control well(polystyrene substrate). At 72 hours, a slight decline was observed inthe growth curves of cells in contact with the three SF patches(microfibers, nanofibers and micro/nano hybrid), which was immediatelyrecovered at 120 hours. It can be concluded that the rate of humanfibroblast proliferation is not disturbed by the presence of threedifferent SF patches.

Human endothelial cells in contact with the three SF biomaterials showeda decline in the degree of proliferation compared to the control. It isworth noting that, as for the test with human fibroblasts, the three SFbiomaterials show almost the same trend in terms of cell proliferationup to 72 hours. However, with human endothelial cells at 120 hours withthe nanofibrous patch they show a further decline of the curve while thehybrid micro/nanofibrous patches had an increase in the measuredmetabolic activity of cells, which is directly linked to cellproliferation.

The genotoxicity test results (FIG. 12) for human fibroblasts showed anincrease in the degree of phosphorylation of H2AX after 72 hours ofincubation. This result is in accordance with the trend of the AlamarBlue® proliferation test, which showed a slight decline in the metabolicactivity of cells at the same incubation time, probably attributable toan adaptation phase of cells cultured on SF biomaterials. At 24 and 120hours, the human fibroblasts cultured on three SF biomaterial showedsignificantly lower levels of phosphorylation compared with controlcells cultured on polystyrene, suggesting that the three SF patches areless genotoxic to these cells than the control substrate. As alreadyobserved with the Alamar Blue® test, human endothelial cells behavedifferently from human fibroblasts: only the microfiber substrateinduces an increase in the phosphorylated form of H2AX at 24 and 120hours, indicating a slightly higher genotoxic effect of this SF patchcompared to the control substrate. On the other hand, the nanofibrouspatch and, most importantly, the micro/nanofibrous hybrid patch displaysthe DNA damage to a lesser extent, indicating a better biocompatibilityin terms of genotoxicity to this type of human cells.

Comment to the Results

As demonstrated by the above tests, the composite materials of thepresent invention have properties that partly reproduce those of theseparate nano- and microfibers, but also new features resulting from thecoupling of the two types of fiber (dynamometric tests, FTIR and DSC).

The chemical analysis results show that the hybrid structure hasessentially the same amino acid composition of the starting microfibrousand nanofibrous parts, characterized by the presence of large amounts ofonly 4 amino acids (glycine+alanine+serine+tyrosine=89% moles), whileall the other amino acids are present in small amounts (approximately11% total moles). It can be concluded that the coupling process does notmodify the chemical structure of fibroin and that the biologicalchemotactic properties of the polymer therefore remains unchanged evenafter coupling.

Moreover, compared to the materials of the prior art, the materials ofthe invention display better adhesion and a more consistent behavior inmechanical tests.

In particular, the SEM images of hybrid structures of the invention(FIGS. 2 and 3) show good adhesion between the parts and the presence ofa continuous polymer film between the fibers of the same two parts,while the similar images of structures of the closest prior art (patentapplication CN 101879330 A, FIG. 4) show a polymer film, which shouldguarantee the adhesion between the parts, which is fragmented andadhering to only one of these.

Tensile tests showed that the composite material of the invention hasunique features in the elongation area between about 10 and 25%, duespecifically to the interactions between the two types of fibers. To thecontrary, the coupled material of the prior art shows a behavior that isthe pure sum of the behaviors of the nano and microfibrous components(FIG. 8-B), demonstrating that the micro and nano components coupledtogether by means of porous material behave as separate phases, eachretaining its inherent properties, without showing anychange/improvement caused by the coupling technique; rather, byproducing a hybrid structure according to the closest prior art, aworsening in the tensile properties of the microfibrous component isobtained, changing from load and elongation at break values of about 63N and 48% to values of 40 N and 30%, respectively.

Similarly, the dynamometric peeling tests of the two layers of thehybrid structures confirmed a much higher adhesion strength between thenano- and microfibrous parts in the case of the present invention thanin the case of the prior art (FIG. 10).

The prior art process therefore does not guarantee the same continuityfeatures between the two parts of the final hybrid structure obtainedwith the process of the present invention: in the case of devicesmanufactured according to the prior art, this may lead to the productionof morphological and mechanical discontinuities among the differentlayers, resulting in loss of the performance and geometriccharacteristics, up to yield and/or collapse of the weaker (e.g.nanofibrous) layers from a mechanical point of view. In stressing useconditions from the mechanical and biological point of view, such asthose that can occur in the progress of an in vivo implantation, thedifferent behavior of the two or more polymer phases that make up thehybrid structure of the prior art could create local stresses of such amagnitude as to interfere with the regenerative processes in progress,especially if the material is exposed to flows of physiological fluids.

The hybrid structures of the invention showed a better in vitrobiological behavior than the individual parts of microfibrous fibroinand of nanofibrous fibroin further enhancing, from the biologicalbehavior point of view, the already good performance levels of micro andnanofibrous parts taken separately: the scaffold performance ofmicrofibroin alone have been described for example in the article “Denovo engineering of reticular connective tissue in vivo by silk fibroinnonwoven materials”, Dal Pra et al., Biomaterials (2005) 26 1987.

The invention claimed is:
 1. Process for the production of a hybridstructure made of microfibers and nanofibers of silk fibroin coupled toone another, which comprises the following steps: a) preparation of oneor more parts made of microfibrous fibroin; b) preparation of one ormore parts made of nanofibrous fibroin; c) treatment of said one or moreparts of nanofibrous fibroin and of said one or more parts ofmicrofibrous fibroin, separately or after coupling, with a solvent forfibroin and/or with a solution comprising fibroin dissolved in thesolvent; c′) if in step c) the nanofibrous and microfibrous parts havebeen treated separately with a solvent for fibroin and/or with asolution comprising fibroin dissolved in a solvent, coupling of saidparts; d) consolidation of the hybrid microfibrous/nanofibrous structureobtained in step c) or in step c′) by thermal treatment at a temperaturebetween 10° C. and 150° C., for a time between 1 minute and 24 hours; e)removal of the solvent by washing with water or a water-alcohol mixtureat a temperature between 10° C. and 100° C., or by evaporation at atemperature between 10° C. and 100° C., wherein the solvent for fibroinor the solvent of the solution comprising fibroin used in step c) isselected from: (i) formic acid, 1,1,1,3,3,3-hexafluoro-2-propanol,trifluoroacetic acid, hexafluoroacetone, N-methylmorpholine N-oxide,ionic liquids, and mixtures thereof, wherein said solvents are pure orin mixture with water; (ii) calcium chloride-ethanol-water mixtures,calcium nitrate-methanol-water mixtures, aqueous solutions of lithiumsalts and; (iii) mixtures among the solvents of (i) and (ii).
 2. Theprocess according to claim 1, further comprising the following steps: f)coupling of two or more hybrid structures produced according to claim 1,obtaining a hierarchically superior hybrid structure; g) repeating stepsc) to e) of claim 1 with said hierarchically superior hybrid structure.3. The process according to claim 1, wherein step a) is carried outusing as starting material microfibrous fibroin in the form of a silkyarn having a count between 10 den and 400 den, scoured previously orsubsequently to the production of said one or more parts of microfibrousfibroin, and wherein said one or more parts of microfibrous fibroin areproduced with a technique selected from weft-warp weaving, knitting,braiding, filament winding and a non-woven fabric production technique.4. The process according to claim 1, wherein step b) is carried out byforce-spinning or electrospinning of a solution of fibroin in a solventselected from formic acid, 1,1,1,3,3,3-hexafluoro-2-propanol,trifluoroacetic acid, ionic liquids, mixtures thereof, wherein saidsolvents are pure or in mixture with water, and wherein theconcentration of fibroin is between 1% w/v and 30% w/v in case thesolvent is formic acid, and between 5% w/v and 40% w/v in case one ormore of said solvents different from formic acid are employed.
 5. Theprocess according to claim 4, wherein said solvent is an ionic liquidselected from 1-ethyl-3-methylimidazolium chloride,1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazoliumacetate and 1-ethyl-3-methylimidazolium glycine.
 6. The processaccording to claim 4, wherein said one or more parts of nanofibrousfibroin are produced by electrospinning, by using a spinneret with anozzle of diameter between 0.01 mm and 10 mm, a distance between saidnozzle and a nanofiber collector between 5 cm and 60 cm, and a potentialdifference between said nozzle and collector between 5 kV and 100 kV. 7.The process according to claim 4, wherein one or more bioactive agentsselected from growth factors, drugs, cells, antibiotics, antivirals,enzymes and vitamins are added to said solution of fibroin.
 8. Theprocess according to claim 1, wherein step c) is carried out bytreatment of said one or more parts prepared in step a) and/or said oneor more parts prepared in step b) with solvent alone, by: immersion ofsaid one or more parts in the solvent for a time between 1 second and240 minutes; or deposition of the solvent by pouring, coating, spraying,electrospray or electrospinning, in an amount between 0.001 and 0.5 mLper 1 cm² of the surface of said one or more parts; or exposure of saidone or more parts to vapors of the solvent for a time between 1 secondand 120 minutes, operating at a temperature between 40 and 80° C. in thecase of parts made with microfibrous fibroin and between roomtemperature and 70° C. in the case of parts made with nanofibrousfibroin.
 9. The process according to claim 1, wherein step c) is carriedout by treatment of said one or more parts prepared in step a) and/orsaid one or more parts prepared in step b) with a solution of fibroin ina solvent, by: immersion of said one or more parts in the solution for atime between 1 second and 240 minutes; or deposition of the solution bypouring, coating, spraying, electrospray or electrospinning, in anamount between 0.001 and 0.5 mL per 1 cm′ of surface of said one or moreparts, operating at a temperature between 40 and 80° C. in the case ofparts made with microfibrous fibroin and between room temperature and70° C. in the case of parts made from nanofibrous fibroin.
 10. Theprocess according to claim 9, wherein said solution contains between 1and 30% w/v fibroin in formic acid, or between 5% and 40% w/v fibroin inone or more solvents selected from 1-ethyl-3-methylimidazolium chloride,1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazoliumacetate or 1-ethyl-3-methylimidazolium glycine or between 5% and 40% w/vfibroin in a mixture of water with one or more solvents selected from1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazoliumchloride, 1-ethyl-3-methylimidazolium acetate or1-ethyl-3-methylimidazolium glycine, wherein said mixture containsbetween 5% and 50% w/w of water.
 11. The process according to claim 1,wherein the evaporation at a temperature between 10° C. and 100° C. ofstep e) is carried out under vacuum.